The binding of 117/1,400 and N6-LS variants to human or rhesus macaque FcγRs was detected by a Biacore T200 surface plasmon resonance system (GE Healthcare) as previously reported (18 (link)). All experiments were performed at 25 °C in HBS-EP+ buffer (10 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid, pH 7.4; 150 mM NaCl; 3.4 mM EDTA; 0.005% [vol/vol] surfactant P20). Human FcγRIIa, FcγRIIb, and FcγRIIIa and rhesus FcγRIIa and FcγRIIIa, diluted at 20 µg/mL in 10 mM sodium acetate, pH 4.5 (or pH 5.0 for human FcγRIIb) were immobilized on Series S CM5 chips by amine coupling, resulting in a density of 2,000 response units. Antibodies were injected through flow cells at different concentrations (ranging from 4,000 to 31.25 nM in 1:2 successive dilutions) at a flow rate of 30 µL/min for 120 s, followed by a 300 s dissociation step. After each assay cycle, the sensor surface was regenerated with a 30 s injection of 25 mM NaOH at a flow rate of 30 µL/min. Background binding to blank immobilized flow cells was subtracted, and sensograms of antibodies binding to FcγRs were generated using Biacore T200 evaluation software (GE Healthcare) with the 1:1 Langmuir binding model.
Biacore t200 evaluation
Manufactured by GE Healthcare
Sourced in Sweden
The Biacore T200 is a label-free biosensor system designed for real-time interaction analysis. The instrument measures biomolecular interactions in real-time without the need for labeling, providing kinetic and affinity data. The Biacore T200 utilizes surface plasmon resonance (SPR) technology to monitor changes in refractive index at the sensor surface, which corresponds to the binding of analytes to immobilized ligands.
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6 protocols using biacore t200 evaluation
1
SPR Analysis of FcγR Binding
2
Kinetics Analysis of A2a Receptor Nanodiscs
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Kinetics analysis was performed at 15 °C using a solution containing 20 mM HEPES pH 7.5, 150 mM NaCl, and 1% dimethyl sulfoxide as running buffer. A2aR nanodiscs were captured onto the active flow cell of the biND5 immobilized chip at 1800 RU. Empty nanodiscs for A2a nanodiscs were captured onto the reference flow cell to reach levels of 1200 RU, considering the mass difference. Kinetics analysis between nanodiscs and ZM241385 were performed using a single-cycle kinetics method. After the startup, which was followed by the solvent correction cycle and dummy cycles, analytes were injected at increasing concentrations. Analyte responses were corrected for buffer responses and subtracted for reference and blank responses. Curve fitting and data analysis were performed using the Biacore T200 Evaluation software (GE Healthcare).
3
Characterization of RNF168 Ubiquitin Binding
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SPR measurements were carried out on a Biacore T200 instrument (GE Healthcare) with HBS-P buffer (10 mM HEPES-Na [pH 7.4], 150 mM NaCl and 0.05% surfactant P20) at 25 °C. Anti-GST antibodies (GE Healthcare) were covalently immobilized on the CM5 sensor chip (GE Healthcare) at a density of about 13,000 resonance units (RU). The GST-fused RNF168 domains were then captured on the sensor chip at a density of 1000–1500 RU. The untagged LRM1–UMI and UDM2ΔC were covalently immobilized on the CM5 sensor chip using amine coupling in 10 mM sodium acetate (pH 4.0 and pH 4.5, respectively). Ub2 species were injected for 60 s at a flow rate of 10 μL per min. Equilibrium dissociation constants (Kd) were computed by fitting steady-state binding level (Req) to a 1:1 interaction model (Req = Concentration × Rmax/(Kd + Concentration) + Offset) using Biacore T200 evaluation software (GE Healthcare). All assays were carried out thrice for each sample. The data are presented as mean ± standard deviation.
4
Quantification of CSB-Ubiquitin Interaction
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SPR measurements were carried out using a Biacore T200 instrument (GE Healthcare) with HBS-P buffer (10 mM HEPES-Na (pH 7.4), 150 mM NaCl and 0.05% surfactant P20) at 25°C. Anti-GST antibodies (GE Healthcare) were covalently immobilized on the CM5 sensor chip (GE Healthcare) at a density of about 13 000 resonance units (RU). The GST-fused CSB WHD was then captured on the sensor chip at a density of 1000–1500 RU. Ub was injected for 60 s at a flow rate of 10 μl per min. Equilibrium dissociation constants (Kd) were computed by fitting steady state binding level (Req) to a 1:1 interaction model using the Biacore T200 evaluation software (GE Healthcare). All assays were carried out thrice for each sample. The data are presented as mean ± standard deviation.
5
Affinity of paeoniflorin and paeonol towards TNF-R1
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Three-fold serial dilutions of paeoniflorin or paeonol (0, 0.14, 0.40, 1.23, 3.70, 11.1, and 33.3 μM) were prepared to determine the affinity constant (KD) of paeoniflorin and paeonol towards TNF-R1. The data were analyzed using Biacore T200 evaluation software (GE Healthcare, Uppsala, Sweden), and the affinity constant was calculated.
6
FOXM1-XST-20 Binding Kinetics
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In this experiment, chemically synthesized FOXM1 (222–360) peptide (Purity (HPLC) >95%) was purchased from Genescript (Piscataway, NJ, USA). The SPR experiment were carried out on a Biacore T200 instrument (GE Healthcare). Briefly, FOXM1 protein was immobilized on a CM5 sensor chip via amine coupling reactions. The sensorgrams were obtained after sequential injection at different concentrations of of XST-20 (from 0.39 to 50 μM). All experiments were carried out at a constant temperature of 25.0 °C. Biacore T200 evaluation software was used with 1:1 binding model fitting for affinity evaluation (GE Healthcare).
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